Despite intense study for decades, major questions regarding the assembly pathway of HIV-1 are still unresolved. In vitro, the protein component of the viral capsid, Gag, is in a monomer-dimer equilibrium in solution, but addition of virtually any nucleic acid leads to highly efficient assembly of virus-like particles (VLPs). Structures of the soluble nucleocapsid domain of Gag binding to nucleic acid have been determined by nuclear magnetic resonance, but it is unclear how this complex proceeds VLP assembly. While cryo-electron microscopic studies of VLPs have elucidated the structure of the capsid domain and flanking regions of Gag, other more flexible Gag domains have remained elusive. In collaboration with the laboratory of Dr. Alan Rein (NCI) we took advantage of the novel biophysical hybrid technique of fluorescence-detected sedimentation velocity developed by us to study the initial interactions between nucleic acid and Gag in solution. Surprisingly, we discovered a heretofore unknown strong dimerization mechanism apparently mediated by a nucleic acid-induced conformational change in Gag. This allosterically induced Gag-Gag interaction is stronger than interactions at any other known Gag-Gag interface, and therefore is likely to constitute the initial oligomerization step leading to assembly of the virion. Through the study of different protein constructs we have localized the new binding interface to the nucleocapsid domain of Gag. From detailed energetic and conformational data we were able to conclude that this interaction promotes complexes with 2:1 protein/nucleic acid composition, most likely in asymmetric conformation where only a single Gag is binding nucleic acid. This suggests a model where high-density Gag patches may form at suitable nucleic acid binding sites, possibly stabilizing assembly intermediates on pathway to capsid assembly.